Pneumonic plague, caused by Yersinia pestis, is highly virulent and frequently causes lethal infection even when diagnosed correctly and antibiotics are given. One (1) of the key virulence features of Y. pestis is the type secretion system and the toxins, called Yops, that are translocated into mammalian cell cytoplasm by the type III secretion system. In mouse model systems of infection, the translocation of Yops is essential for productive infection of Yersinia in lungs. We have generated a reporter system in which Yop translocation signals of YopE are fused to a gene, b-lactamase, which encodes TEM. When TEM is delivered into mammalian cells, it cleaves a fluorescent substrate, CCF2, such that CCF2 emits light at a different wavelength. We can easily detect translocation of YopE-TEM fusion proteins by loading HEp-2 cells withCCF2-AM and detecting the 450 versus 520 emission spectrum of cells with by fluorescence microscopy. We propose to adapt this technology to use in a high through-put screen to identifying small molecular inhibitors of translocation of Yops. Ultimately, CCR-2 loaded HEp-2 cells will be infected with Yersinia strains expressing YopE-TEM fusion proteins in the presence of small molecules. The ratio of 460 versus 520 emissions will be read in a Fluorescence ELISA plate reader. Cells exposed to compounds which inhibit translocation should fluoresce at 520. These compounds will'be tested in several secondary screens to confirm that the compounds inhibit type III secretion and translocation processes. Such inhibitors should provide a novel class of therapeutics against pneumonic plague as well as other pathogens that use type III secretion systems to overcome host defenses, including other pathogens on the Priority pathogens list such as Salmonella, and Y. enterocolitica. Potentially these molecules could be used either prophylatically during an outbreak of pneumonic plague or after initial signs of infection are apparent. In addition, these inhibitors will be valuable tools to probe the structure and function of type III secretion systems encoded by Yersinia species and other Gram-negative bacterial pathogens.